Compare And Contrast Transcriptional Regulation In Eukaryotes And Bacteria

Compare and contrast transcri ptional regulation in eukaryotes and bacteria Thesis Bacteria and eukaryotes employ various methods of transcri ptional regulation in order to control the level of gene expression. Some methods are unique to bacteria, some are unique to eukaryotes and some are shared between both. It appears that the regulation of RNA polymerase and modification of DNA and its packaging molecules are the two main mechanisms in transcri ptional regulation, despite their different regulatory machines.

Regulation of transcri ptional initiation Operons are uniquely found in bacteria. They are the assembly of promoter, operator and structural genes. In E. coli, ara operons provide both positive and negative control depending on the concentration of arabinose present (Schleif, 2003). When arabinose molecule is in abundance, it forms a complex with AraC protein and binds to the araI site. cAMP-CAP complex also binds to the araI site. This recruits the RNA polymerase to transcribe the structural gene used in arabinose catabolism, activating transcri ption. When arabinose is in low concentration, it does not bind to the AraC protein. In this conformation, AraC protein binds to both the araI and araO site in a hairpin loop so RNA polymerase cannot bind to DNA and transcri ption is repressed (Schleif, 2003). Eukaryotes do not have operons so activators and enhancers are the main focus of transcri ptional regulation in the initiation step. cAMP can be used as an eukaryotic regulator. cAMP response elements are enhancers (regulatory sequences) located upstream of structural genes. cAMP response element binding protein (this is an activator) binds to CRE when it is phosphorylated by protein kinase A (PKA is activated by the binding of cAMP to the regulatory units due to extracellular signalling via G proteins which lead to the formation of cAMP from ATP by adenylyl cyclase). CREB binding protein is a co-activator. It binds to CREB on one side and RNA polymerase on the other to allow protein-protein interactions between CREB and RNA polymerase as the DNA forms a U-bend (this process is DNA looping) (Altarejos & Montminy, 2011). Initially RNA polymerase could not bind to the promoter region effectively however, in the presence of co-activator it is able to start transcri ption. Sometimes the addition of specific transcri ption factors to the operator allows further enhancement of RNA polymerase and transcri ption proceeds. Lac operon is a negative inducible control in bacteria. Lactase is expressed when lactose (which is an inducer) binds to the allosteric site in the repressor (coded for by lacI which is a regulatory sequence upstream of the lac operon) and induces a conformational change in the repressor protein. It can no longer bind to the operon and RNA polymerase is allowed to initiate transcri ption (Moulton & Ed.D, 2004). Gene expression via operons is unique in bacteria however in the sense that it is a cis-acting sequence, it is similar to the cis-acting sequences in eukaryotes. For example, the promoter sequences upstream of the structural gene coding for thymidine kinase (Cooper, 2000) contain two GC boxes and a CCAAT box. They recruit specific transcri ption factors which in turn recruit RNA polymerase to initiate transcri ption. Could the cis-acting sequences in eukaryotes be of bacterial origin? To determine this, molecular analysis should be conducted on a wide range of cis-acting sequences in both bacteria and eukaryotes which regulate similar structural gene. And comparisons need to be made to determine their origins. Attenuation Attenuation occurs in the trp operon in E. coli during the early stages of elongation in bacteria. In the structural gene, there is a leader region which is transcribed by RNA polymerase and translated by ribosome concurrently. When tryptophan is in abundance, transcri ption will stop due to a terminating loop forming between sequences three and four. When there is a lack of tryptophan, a terminating loop will form between sequences two and three (Das, et al., 1982). This alternative structure does not bring about the termination of transcri ption. The formation of a terminating loop depends on the speed of the ribosome. If the ribosome stalls at the first sequence in the leader region (where there is two adjacent tryptophan codons) due to a lack of tryptophan tRNA is be able to bring, a non-terminating loop will form. If the ribosome does not stall, a terminating loop will form. Attenuation is a regulatory control that is not present in eukaryotes because it requires transcri ption and translation to occur simultaneously. Eukaryotic cells are compartmentalised so translation cannot be coupled to transcri ption. Histone and DNA modification (epigenetics) In eukaryotes, histone tail phosphorylation on serine residues effects the expression of different genes depending on the position of the targeted serine. For example, the phosphorylation of certain serines on the H3 subunit regulates epidermal growth factor (EGF) (Duncan, et al., 2006) whereas H3S10ph and H2BS32ph lead to the expression of proto-oncogenes (Rossetto, et al., 2012). Sometimes phosphorylation is coupled to the acetylation in the nucleosome (Zhang & Reinberg, 2001). For instance, the phosphorylation of H3S10 promotes the acetylation of H3K9ac and K14ac where they both induce transcri ptional activation (Rossetto, et al., 2012). Acetylation is covalent modification which is controlled by histone acetyltransferase. The DNA backbone is negatively charged due to the phosphate groups. This causes it to bind tightly to the lysine residues on the histone tails. Once acetylated, the charge of the N terminus of certain lysine residues is removed, reducing the histone-DNA interaction. As a result, the DNA is in its relaxed state (euchromatin) and the desired gene sequence become easier for transcri ptional machines to access the DNA template strand. Acetylation also serves as signals for the binding of trans-binding factors. Many regulation factors (e.g. activation factors) with bromodomains interact with acetylated lysine residues (Zeng & Zhou, 2002). In histone modification both phosphorylation and acetylation increase gene expression in transcri ption. Histone modification is not possible in bacteria because histones are absent. Bacterial DNA compaction uses DNA binding proteins to form looped domains so DNA binding proteins in bacteria shows sequence homology and are functionally similar to histones in eukaryotes. DNA binding protein H-NS are global bacterial transcri ptional regulators (they act as repressors for a wide range of gene sequences) (Schrödera & Wagner, 2000) and are paralogous to StpA proteins (Atlung & Ingmer, 1997). The proposed model shows DNA binding protein H-NS to be a dimer. H-NS binds to the promoter sequence in bacterial DNA and dimerises to compact DNA to prevent RNA polymerase from binding. This method of regulation requires the assembly of homomeric proteins (this process is self-assembling so it happens at equilibrium). H-NS proteins also regulate transcri ptional elongation by slowing down the action of RNA polymerase while Rho terminates it (Peters, et al., 2012). Thus functionally, H-NS and Rho show cooperative action. This combined action along with NusG is analogous to Sen1-Nrd1-Nab3 and nucleosome systems in suppression of transcri ption in eukaryotes (Peters, et al., 2012). DNA methylation occurs in both bacteria and eukaryotes to modulate gene expression. Eukaryotic methylation of cytosine bases attract MeCP2 (a protein) which works as a part of a complex with deacetylase to remove the acetyl groups on histones (Phillips, 2008). The histones become negatively charged once again. DNA binds tightly around the octameric histone and ultimately silencing gene expression by limiting the accessibility of DNA. Bacterial methylation is normally used in DNA restriction-modification systems but it can also alter the level of gene expression during transcri ption (Casadesús & Low, 2006). Adenine methylation of the GATC in the 5'noncoding sequences controls the regulatory proteins binding to the promoter sequence in E. coli (Kahramanoglou, et al., 2012) (Moulton & Ed.D, 2004). Methylation of DNA in other sequences can also directly affect the availability of the promoters RNA polymerase bind to. So even though methylation occurs in both bacteria and eukaryotes, the functions of methylated bases are different.

Conclusion Both eukaryotic and bacterial transcri ptional regulation focuses on the initiation step of transcri ption although there are also regulations in the subsequent steps. Operons are used in bacteria whereas enhancers and activators are used in eukaryotes to modulate transcri ptional initiation. Modification of DNA and packaging molecule occurs in both but the mechanisms are very different - perhaps the notion of DNA modification evolved independently in bacteria and eukaryotes. (1294 words)

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